An analysis of specific mRNA levels in a given cell provides insight into the function and differentiative state of that particular cell at any point in its life cycle. However, mRNA levels do not always provide an accurate portrayal of a cell's functional state. It is the translated products of these mRNAs, such as the receptors, ion channels, enzymes, and structural proteins of the cell, that determine its function.
Techniques currently used to detect proteins are based on various types of immunoassays, such as ELISA, immunohistochemistry and radioimmunoassay, which utilize antibodies specific for the protein of interest. These immunoassays, while useful, are limited by the sensitivity of the detection of the antibody. Standard labeling methods include fluorescence, radioisotopes, and enzymes such as peroxidase and phosphatase. In addition, secondary antibodies are oftentimes biotinylated to increase their sensitivity. Still, these techniques are often not capable of detecting small amounts of a particular antigen. Furthermore, these types of techniques are not feasible for detection of a specific protein from a particular cell.
Electrophysiological techniques can detect protein from a specific cell. However, applications are limited to the monitoring of ion channel functioning, as well as the functioning of other receptors or proteins which are coupled to channels. It is difficult to detect the small amounts of other proteins that do not directly couple to ion channels, which techniques such as expression profiling and immunohistochemistry indicate are present and potentially regulated within an individual cell.
Recently, Sano et al., 1992 Science, 258:120-122, described an antigen detection technique termed immuno-polymerase chain reaction (immuno-PCR). This procedure provides an extremely sensitive method to detect proteins. In immuno-PCR, a linker molecule with bi-specific binding affinity for DNA and antibody is used to attach a marker DNA molecule specifically to an antigen-antibody complex, thus resulting in the formation of a specific antigen-antibody-DNA conjugate. The attached marker DNA can be amplified by PCR with the appropriate primers. The presence of specific size PCR products demonstrates that marker DNA molecules are attached specifically to antigen-antibody complexes thereby indicating the presence of antigen. As described by Sano et al. 1992, antigen is immobilized on the surface of microtiter plates and then detected by immuno-PCR. Using this technique, an approximately 10.sup.5 increase in sensitivity over an alkaline phosphatase conjugated ELISA was obtained. Sensitivity advantages of immuno-PCR have subsequently been confirmed in assays for mouse anti-lipoprotein IgG (Ruzicka et al., 1993 Science, 260:698-699); a human proto-oncogene protein (Zhou et al., 1993 Nucleic Acid Res., 21:6038-6039); and tumor necrosis factor alpha (Sanna et al., 1995 Proc. Natl. Acad. Sci., 92:272-275).
More recent reports have described advancements in immuno-PCR technology. For example, Joerger et al., 1995 Clin. Chem., 41 (9):1371-1377) demonstrate that double-stranded DNA labels can be directly attached to antibodies thus allowing conjugate reagents to be prepared before the assay. Suzuki et al., 1995 Jpn. J. Cancer Res., 86:885-889, describe a method called double determinant immuno polymerase chain reaction (double-determinant immuno-PCR) which utilizes two monoclonal antibodies, in which the antigens are sandwiched, and a specific DNA molecule is used as a marker. Instead of the antigen itself, the first monoclonal antibody to bind the circulating antibody is immobilized, the biotinylated second monoclonal antibody is bound to the antigen and free streptavidin is used to attach a biotinylated DNA to the second monoclonal antibody. The biotinylated DNA complexed with antigen-antibody-streptavidin is amplified by PCR, and the products analyzed by Southern blot analysis. While this technique has provided advantages over traditional methods of protein detection, such as an increase in sensitivity, there still exist several notable limitations. For example, the use of the polymerase chain reaction is not quantitative. While PCR can be used to "amplify" a marker sequence to detect rarely occurring proteins, this amplification is not quantitative for &gt;10-fold differences in antigen concentration. Thus, there is no direct correlation between the amount of signal and the amount of protein present. The immuno-PCR method also has inherent limitations that make it difficult to detect the presence of antigen in a particular cell. This is particularly relevant when antigen expressed in a specific cell type, such as a neuron, is being assayed. Further, these detection techniques can only assay protein present in solution or tissue, which is often a mixture of cell types.
Accordingly, there exists a need for an easily adaptable, sensitive detection method that is semi-quantitative and that can provide an accurate protein profile for a specific cell.